1. Field of the Invention
A method for coordinate expression in a single cell, in vivo, of exogenous genes via introduction into the tissue of a vertebrate of polycistronic polynucleotide constructs is described. The method results in production of immune responses against the products produced as a result of expression of the exogenous genes. The method and polynucleotide constructs of this invention may be used in a vertebrate to generate immune responses against antigenic epitopes expressed by a single cell. The coordinate expression results in improved expression of gene products which may be otherwise poorly expressed. It also results in improved cellular immune responses due to provision of T-cell stimulatory signals by the same cell expressing T-cell antigens. Polynucleotide constructs encoding human immunodeficiency virus (HIV) antigens exemplify one embodiment of the method.
2. Background of the Invention
A major challenge to the development of vaccines against viruses, particularly viruses with a high rate of mutation such as HIV, against which elicitation of neutralizing and protective immune responses is desirable, is the diversity of the viral envelope proteins among different viral isolates or strains. Because cytotoxic T-lymphocytes (CTLs) in both mice and humans are capable of recognizing epitopes derived from conserved internal viral proteins and may be important in the immune response against viruses, efforts have been directed towards the development of CTL vaccines that elicit heterologous protection against different viral strains.
CD8+ CTLs kill virally-infected cells when their T cell receptors recognize viral peptides associated with MHC class I molecules. These peptides are derived from endogenously synthesized viral proteins. Thus, by recognition of epitopes from conserved viral proteins, CTLs may provide cross-strain protection. Peptides capable of associating with MHC class I for CTL recognition originate from proteins that are present in or pass through the cytoplasm or endoplasmic reticulum. Exogenous proteins which enter the endosomal processing pathway (as in the case of antigens presented by MHC class II molecules) are not usually effective in generating CD8+ CTL responses.
Efforts to generate CTL responses have used replicating vectors to produce the protein antigen within the cell or have introduced peptides into the cytosol. These approaches have limitations that may limit their utility as vaccines. Retroviral vectors have restrictions on the size and structure of polypeptides that can be expressed as fusion proteins while maintaining the ability of the recombinant virus to replicate. Further, the effectiveness of vectors such as vaccinia for subsequent immunizations may be compromised by immune responses against the vectors themselves. Also, viral vectors and modified pathogens have inherent risks that may hinder their use in humans [R. R. Redfield et al., New Engl. J. Med. 316, 673 (1987); L. Mascola et al., Arch. Intern. Med. 149, 1569 (1989)]. Furthermore, the selection of peptide epitopes to be presented is dependent upon the structure of an individual's MHC antigens; thus, peptide vaccines may have limited effectiveness due to the diversity of MHC haplotypes in outbred populations.
Benvenisty, N., and Reshef, L. [PNAS 83, 9551–9555, (1986)] showed that CaCl2-precipitated DNA introduced into mice intraperitoneally (i.p.), intravenously (i.v.) or intramuscularly (i.m.) could be expressed. Intramuscular injection of DNA expression vectors in mice results in the uptake of DNA by the muscle cells and expression of the protein encoded by the DNA [J. A. Wolff et al., Science 247, 1465 (1990); G. Ascadi et al., Nature 352, 815 (1991)]. The plasmids were maintained episomally and did not replicate. Subsequently, persistent expression has been observed after i.m. injection in skeletal muscle of rats, fish and primates, and cardiac muscle of rats. The technique of using nucleic acids as therapeutic agents was reported in WO90/11092 (4 Oct. 1990), in which naked polynucleotides were used to vaccinate vertebrates.
It is not necessary for the success of the method that immunization be intramuscular. Thus, Tang et al., [Nature, 356, 152–154 (1992)] disclosed that introduction of gold microprojectiles coated with DNA encoding bovine growth hormone (BGH) into the skin of mice resulted in production of anti-BGH antibodies in the mice. Furth et al., [Anal. Biochem. 205, 365–368, (1992)] showed that a jet injector could be used to transfect skin, muscle, fat, and mammary tissues of living animals. Methods for introducing nucleic acids was recently reviewed by Friedman, T., [Science, 244, 1275–1281 (1989)]. Robinson et al., [Abstracts of Papers Presented at the 1992 meeting on Modern Approaches to New Vaccines, Including Prevention of AIDS, Cold Spring Harbor, p92] reported that i.m., i.p., and i.v. administration of avian influenza DNA into chickens provided protection against lethal challenge. However, Robinson et al. did not disclose which avian influenza virus genes were used. In addition, only H7 specific immune responses were alleged; the induction of cross-strain protection was not discussed. Intravenous injection of a DNA:cationic liposome complex in mice was shown by Zhu et al., [Science 261:209–211 (9 Jul. 1993); see also WO93/24640, 9 Dec. 1993] to result in systemic expression of a cloned transgene. Recently, Ulmer et al., [Science 259:1745–1749, (1993)] reported on the heterologous protection against influenza virus infection by injection of DNA encoding influenza virus proteins.
The need for specific therapeutic and prophylactic agents capable of eliciting desired immune responses against pathogens and tumor antigens is achieved by the instant invention. Of particular importance in this therapeutic approach is the ability to induce T-cell immune responses which can prevent infections or disease caused by virus strains which are heterologous to the strain from which the antigen gene was obtained. This is of significance with HIV, since HIV mutates rapidly, and because many virulent isolates have been identified [see, for example, LaRosa et al., Science 249:932–935 (1990), identifying 245 separate HIV isolates].
In response to this diversity, researchers have attempted to generate CTLs by peptide immunization. Thus, Takahashi et al., [Science 255:333–336 (1992)] reported on the induction of broadly cross-reactive cytotoxic T cells recognizing an HIV envelope (gp160) determinant. They recognized the difficulty in achieving a truly cross-reactive CTL response and suggested that there is a dichotomy between the priming or restimulation of T cells, which is very stringent, and the elicitation of effector function, including cytotoxicity, from already stimulated CTLs.
Wang et al., [P.N.A.S. USA 90:4156–4160 (May, 1993)] reported on elicitation of immune responses in mice against HIV by intramuscular inoculation with a cloned, genomic (unspliced) HIV gene. The level of immune response achieved was low, and the system utilized portions of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) promoter and portions of the simian virus 40 (SV40) promoter and terminator. SV40 is known to transform cells, possibly through integration into host cellular DNA. Therefore, unlike the system described herein, the system described by Wang et al. may be inappropriate for administration to humans. In addition, the DNA construct of Wang et al. contains an essentially genomic piece of HIV encoding contiguous Tat/REV-gp160-Tat/REV coding sequences (FIG. 1). As is described in detail below, this is a suboptimal system for obtaining high-level expression of the gp160. One drawback is that the expression of Tat has been recognized to play a contributory role in the progression of Kaposi's Sarcoma, [Y. N. Vaishav and F. W. Wong-Staal, An. Rev. Biochem. (1991)].
WO 93/17706 describes a method for vaccinating an animal against a virus, wherein carrier particles were coated with a gene construct and the coated particles are accelerated into cells of an animal. In regard to HIV, essentially the entire genome, minus the long terminal repeats, was proposed to be used. That method may represent a substantial risk for recipients. Constructs of HIV should, in general, contain less than about 50% of the HIV genome to ensure safety of the vaccine. Thus, a number of problems remain if a useful human HIV vaccine is to emerge from the gene-delivery technology.
The instant invention uses known methods for introducing polynucleotides into living tissue to induce expression of proteins. This invention provides a immunogen for introducing HIV and other proteins into the antigen processing pathway to efficiently generate HIV-specific CTLs and antibodies. The pharmaceutical is effective as a vaccine to induce both cellular and humoral anti-HIV and HIV neutralizing immune responses. The instant invention addresses some of the problems by providing polynucleotide immunogens which, when introduced into an animal, direct the efficient expression of HIV proteins and epitopes without the attendant risks associated with those methods. The immune responses generated are effective at recognizing HIV, at inhibiting replication of HIV, at identifying and killing cells infected with HIV, and are cross-reactive against many HIV strains. Therefore, this invention provides a useful immunogen against HIV. The invention also provides polynucleotide constructs which enable the co-expression, in vivo, of more than one gene-product in a single cell. This is demonstrated with an HIV gene expression system in which the expression of a first gene is dependent on the co-expression in the same cell of a second gene product. By virtue of the success of achieving this co-expression in vivo, it is now predictable that this type of polynucleotide construct may be applied to co-expression in vivo of many combinations of gene products, including but not limited to viral antigens other than HIV related antigens, carcinoma-associated antigens, and immunomodulatory or immunostimulatory gene products.